Abstract BRAF kinase is frequently found mutated in human tumors and in the majority of melanomas. RAF inhibitors vemurafenib and dabrafenib improved survival of melanoma patients with BRAF(V600E) tumors. Unfortunate- ly, responses are usually temporary, followed by development of resistance, most commonly due to ineffective inhibition of RAF and reactivation of ERK signaling in the presence of the drug. In addition, RAF inhibitors in- duce second site tumors, due to RAS-dependent paradoxical activation of RAF and downstream ERK signaling in normal cells. Outside of melanoma, RAF inhibitors showed limited efficacy in patients colorectal and thyroid BRAF(V600E) tumors, also due to ineffective inhibition of RAF/ERK signaling in these tumors. More recently, combinations of RAF and MEK inhibitors showed improved efficacy compared to RAF inhibitor monotherapy, but resistance eventually emerges as well. Resistance mechanisms identified to the RAF/MEK inhibitor combi- nation are similar to the ones identified for RAF inhibitor monotherapy, suggesting that ineffective inhibition of RAF is a critical limiting factor in both contexts. There is thus a pressing need for improved therapeutic strate- gies targeting oncogenic BRAF, such that durable responses and minimal side effects can be achieved. Our previous work showed that regulation of BRAF kinase by dimerization determines both the development of re- sistance to currently used RAF inhibitors and inhibitor-induced RAF paradoxical activation, but the underlying mechanisms remain incompletely understood. Recently, next generation RAF inhibitors with different structural and biochemical properties have entered preclinical and clinical development, but the most appropriate clinical context for their use is unknown. The goal of this proposal is to accomplish a detailed understanding of the mechanisms governing the targeting of oncogenic BRAF by small molecules inhibitors and to use this knowledge in order to design more effective RAF inhibitor-based therapeutic strategies. More specifically, we will 1) characterize the mechanistic basis of resistance to RAF inhibitors due to BRAF dimerization by linking the conformational changes induced by inhibitor binding to BRAF to the biochemical effects of the inhibitor, 2) gain a detailed understanding of the biochemical mechanism of paradoxical RAF activation by inhibitors in cells with wild-type BRAF and 3) identify effective RAF inhibitor-based therapeutic strategies for tumors with dimeric BRAF that would overcome RAF dimer-mediated intrinsic or acquired resistance to RAF inhibitors. The mech- anistic insights gained by the proposed experiments will enable the rational design of more effective small mol- ecule inhibitors and RAF inhibitor-based therapeutic strategies targeting oncogenic BRAF signaling, with mini- mal side effects and prolonged time to resistance.